The broad goal of this project is to understand how dynamic microtubules are controlled during cell division to build the mitotic spindle and to segregate chromosomes with high fidelity. This project is designed to investigate the mechanism by which mitotic microtubule motor proteins of the kinesin family regulate microtubule dynamics in order to facilitate mitotic spindle assembly, chromosome segregation and long-term stability of chromosome number. Our approach is interdisciplinary, ranging from single molecule biophysical analyses using total internal reflection microscopy (TIRF) and purified components to cellular approaches using high resolution quantitative live cell imaging, mutant constructs and siRNA depletions. We will use TIRF microscopy of live microtubules at physiological temperature and buffer conditions to evaluate the effect of mitotic kinesins on microtubule assembly and disassembly. Armed with an understanding of the activity the motor provides to microtubules in isolation we will transfer our studies to live mitotic cells. Using hig resolution imaging, selective depletions of kinesins and mutant analogs we will evaluate how kinesin motor activity is used within the mitotic spindle. This is essential basic knowledge which will guide the development of drugs targeted to mitotic kinesins for use in cancer treatment. Finally, we have preliminary evidence that small alterations in microtubule dynamics that do not have a profound impact on cell division in the short term have significant effects on long-term chromosome instability. Furthermore, known tumor suppressor genes which have not previously been linked to microtubules may exhibit unexpected direct or indirect effects on microtubule dynamics. We have a simple visual screen that will provide us with a panel of genes implicated in microtubule dynamics alterations and, by extension, chromosome instability. This will assist in molecular characterization of cancers and targeted drug development. Using these approaches we will learn how motile kinesins target to and regulate polymer assembly and disassembly at microtubule ends and what effect alterations in microtubule dynamics has on short-term spindle assembly and chromosome segregation. This project is designed to evaluate the effect that small changes in microtubule dynamics in the mitotic spindle has on chromosome instability and, in this way, understand the mechanistic forces underlying tumor progression.

Public Health Relevance

During cell division chromosomes must be segregated to each daughter cell with perfect fidelity to avoid the alterations in chromosome numbers characteristic of cancer cells. This process is critically dependent on dynamic polymers within the cell called "microtubules" and cells have evolved numerous motor molecules that regulate microtubule assembly and disassembly. By investigating the mechanism of action of these regulators we will uncover new, tissue-specific targets for cancer therapies and increase our understanding of the molecular events that drive cell division in normal and cancer cells.